Download An rpoB signature sequence provides unique resolution for the

International Journal of Systematic and Evolutionary Microbiology (2011), 61, 170–183
DOI 10.1099/ijs.0.019018-0
An rpoB signature sequence provides unique
resolution for the molecular typing of cyanobacteria
Virginie Gaget,13 Simonetta Gribaldo2 and Nicole Tandeau de Marsac14
Correspondence
1
Virginie Gaget
2
[email protected]
Institut Pasteur, Unité des Cyanobactéries; URA CNRS 2172, 75015 Paris, France
Institut Pasteur, Unité de Biologie Moléculaire du Gène Chez les Extrêmophiles; 75015, Paris,
France
The use of morphological characters for the classification of cyanobacteria has often led to
ambiguous strain assignment. In the past two decades, the availability of sequences, such as
those of the 16S rRNA, nif, cpc and rpoC1 genes, and the use of metagenomics, has steadily
increased and has made the reconstruction of evolutionary relationships of some cyanobacterial
groups possible in addition to improving strain assignment. Conserved indels (insertions/
deletions) are present in all cyanobacterial RpoB (b subunit of RNA polymerase) sequences
presently available in public databases. These indels are located in the Rpb2_6 domain of RpoB,
which is involved in DNA binding and DNA-directed RNA polymerase activity. They are variable in
length (6–44 aa) and sequence, and form part of what appears to be a longer signature sequence
(43–81 aa). Indeed, a number of these sequences turn out to be distinctive among several strains
of a given genus and even among strains of a given species. These signature sequences can thus
be used to identify cyanobacteria at a subgenus level and can be useful molecular markers to
establish the taxonomic positions of cyanobacterial isolates in laboratory cultures, and/or to
assess cyanobacterial biodiversity in space and time in natural ecosystems.
INTRODUCTION
Cyanobacteria represent a monophyletic group of oxygenic
photosynthetic bacteria within the Eubacteria. Since their
appearance between 2.45 and 2.32 billion years ago
(Rasmussen et al., 2008), the cyanobacteria have developed
a number of adaptive strategies and thus form a very
diverse group of prokaryotes. Their classification has been
established based on morphological and physiological
criteria under the International Code of Botanical
Nomenclature (ICBN) (Greuter et al., 2000). In 1978,
having recognized their truly prokaryotic nature, Stanier
et al. (1978) proposed that their nomenclature be governed
by the provisions of the International Code of Nomenclature of Bacteria (ICNB) (Lapage et al., 1992). Although
efforts to harmonize the use of both Codes and to achieve a
consensus nomenclature have been made over the past
25 years to further integrate cyanobacteria into the
Bacteriological code, the names of only a very limited
number of taxa have been validly published under the
3Present address: United Water, 180 Greenhill Road, 5063 Parkside,
SA, Australia.
4Present address: Laboratoire de Chimie Bactérienne, UPR CNRS
9043, Université d’Aix-Marseille, 13402 Marseille cedex 20, France.
Abbreviations: ML, maximum-likelihood; MP, maximum-parsimony; NJ,
neighbour-joining.
Supplementary data are available with the online version of this paper.
170
Bacteriological Code (Trüper, 1986; Oren, 2004). This dual
nomenclature has resulted in the use of multiple names for
the same organism, causing considerable confusion in the
literature. Therefore, cyanobacterial taxonomy and strain
assignment still remains an important issue in the scientific
community.
Since the development of molecular biology methods, the
taxonomy of many bacterial taxa has been challenged, and
the morphological traits employed for the description of
cyanobacteria are not sufficient to unambiguously identify
or describe a new isolate, resulting in a classification that is
not always phylogenetically coherent (Honda et al., 1999;
Ishida et al., 2001; Wilmotte & Herdman, 2001). On the
other hand, sequence analysis of the 16S rRNA gene, largely
used to establish the phylogenetic relationships among
cyanobacteria, has only in some cases led to the description of phylogenetically coherent taxa (Giovannoni et al.,
1988; Turner et al., 1999; Wilmotte & Herdman, 2001).
Therefore, additional genetic markers, used alone or in
combination with morphological criteria, are often necessary to resolve some specific nodes or taxa (Rajaniemi
et al., 2005; Suda et al., 2002; Tanabe et al., 2007).
The gene encoding the RNA polymerase b subunit (rpoB)
has been shown to be a reliable genetic marker for
phylogenetic analyses for many bacterial phyla (Case
et al., 2007; Hong et al., 2004; Salerno et al., 2007;
Volokhov et al., 2007). Interestingly, an indel (insertion/
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rpoB signature sequence for cyanobacterial typing
deletion) was noticed in the rpoB sequences of
Nostocaceae, but was not discussed further (Rajaniemi
et al., 2005). Indels have been used as phylogenetic
characters to assess evolutionary relationships among many
bacterial and archaeal phyla (Gupta, 1997, 1998) and
recently among cyanobacterial taxa (Gupta, 2003, 2009).
However, as indels often correspond to protein loops, they
can easily be lost independently in evolutionarily unrelated
sequences and thus give rise to misleading convergences.
This phenomenon of convergence limits the use of indels for
the investigation of large-scale evolutionary relationships.
However, in combination with molecular phylogenies,
indels may resolve some specific nodes, and also may be
useful for sequence comparisons at a genus or species level.
In the present study, we sequenced part of the rpoB gene
from different strains of cyanobacteria belonging to the
genus Oscillatoria and investigated the presence and
distribution of a specific signature (including an indel) in
their RpoB sequences, as well as in publicly available RpoB
sequences from 118 additional cyanobacterial strains.
Comparative analyses of the amino acid sequences of these
signatures reveal that they might be a very useful tool for
cyanobacterial screening and assignment at a subgenus
level.
METHODS
Strains and culture conditions. The 16 Planktothrix and
Oscillatoria strains used in this study (indicated in bold in
Supplementary Table S1, available with the online version of this
paper) were supplied by the Pasteur Culture Collection of
Cyanobacteria (PCC). All strains were incubated at 25 uC under
white light (Osram Universal White) with a photosynthetic photon
flux density (PPFD) of 30 mmol quanta m22 s21 (measured with a
LICOR LI-185B quantum/radiometer/photometer equipped with a
LI-190SB quantum sensor). All strains were maintained in liquid BG11 medium (Rippka, 1988) containing 0.4 mM Na2CO3, except
strains PCC 10106 and PCC 10110, for which the concentration of
NaNO3 in the BG-11 medium was reduced to 2 mM.
DNA extraction, amplification and sequencing. Aliquots (2 ml)
of liquid cultures were centrifuged at 12 000 g for 10 min at 20 uC
and washed twice with sterile MilliQ water. The final pellets were
resuspended in 200 ml sterile MilliQ water and frozen in liquid
nitrogen. Lysates of the frozen samples were prepared by a minimum
of ten alternating cycles of thawing at 100 uC for 5 min and refreezing
in liquid nitrogen. For Planktothrix and Oscillatoria strains, specific
primers RPObF1 (59-AGGAATTCACCACCACAACT-39) and
RPObR1 (59-ACCATCGGCTAATACCTG-39) were designed to
amplify a 600 bp region. For the 16S rRNA total amplification, the
forward primer A2 (59-AGAGTTTGATCCTGGCTCAG-39; Iteman
et al., 2002) and the 16SR1 primer (59-GGTCTCCCTAAAAGGAGGTG-39) located respectively at the beginning of the 16S rRNA and in
the internal transcribed spacer (ITS) were used. The DNA sequences
used for primer design were collected from the NCBI (National
Center for Biotechnology Information) database (www.ncbi.nlm.nih.
gov). New primers were designed with Primer 3 v.0.4.0 (http://frodo.
wi.mit.edu/) using the sequences of Planktothrix sp. NIVA-CYA 126
and NIVA-CYA 127 (AJ628133, AJ783334). The PCR mixtures, in a
final volume of 50 ml, contained 5 ml lysate, 5 ml 106 PCR buffer,
1.5 ml 50 mM MgCl2, 1.5 ml 2.5 mM deoxyribonucleotides mix,
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1.25 ml each primer diluted to 0.1 mg ml21 (MWG Biotech) and 5 ml
5 U Taq DNA polymerase ml21 (Invitrogen). After an initial step
consisting of 5 min at 95 uC, 35 cycles of amplification were
performed; each amplification cycle consisting of 30 s at 95 uC, 30 s
at 52 uC and 1 min at 72 uC. A final elongation step was carried out
for 5 min at 72 uC. All DNA amplifications were performed in a
GeneAmp PCR System 9700 thermocycler (Applied Biosystems). A
total of 10 ml of each sample was analysed by electrophoresis on 2 %
(w/v) agarose (Litex FMC) gels (200 ml) in 16 Tris-borate/EDTA
buffer (pH 8.0). For visualization of the products, the gels were
stained with 200 ml ethidium bromide (0.7 mg ml21). After amplification, fragments of interest were sent to MWG Biotech for
sequencing using cycle sequencing technology (dideoxy chain
termination/cycle sequencing) on ABI 3730XL sequencing machines.
Sequence retrieval and alignment. Except for the sequences of the
16 strains established in the present study, the rpoB and 16S rRNA
gene sequences of 118 other cyanobacterial strains were retrieved
from the NCBI database (Table S1). Both the nucleotide sequences
and their corresponding amino acid sequences were aligned with
CLUSTAL_X v.1.83 (Jeanmougin et al., 1998). Multiple alignments were
edited with GeneDoc v.2.6.002 (Nicholas et al., 1997) and
ambiguously aligned positions were removed using Gblocks v.0.91b
with default parameters (Castresana, 2000).
Phylogenetic analyses. Phylogenetic trees were calculated on
nucleotide alignments by using three methods: neighbour-joining
(NJ), maximum-parsimony (MP) and maximum-likelihood (ML).
NJ (Jukes–Cantor model) and MP trees were calculated by using
MEGA4 (Tamura et al., 2007). ML trees were computed with Phyml
v.2.4.1 (Guindon & Gascuel, 2003) and the HKY (Hasegawa–
Kishino–Yano) model of nucleotide substitution (Hasegawa et al.,
1985) using one category of substitution rate. For each method, the
robustness of each branch was estimated by non-parametric bootstrap
analysis (100 replicates) using the two programs cited above.
Sequences from strains of each monophyletic group were extracted
from the general alignment, and sequences of strains among this
group were independently realigned, giving a new tree. This operation
was repeated until there was no further resolution in the resulting
tree. These consecutive trees are called tier trees in this study.
Signature sequence localization. The position of the indel within
the rpoB gene was visualized using SMART (http://smart.emblheidelberg.de/). The position of the indel and its impact on the
three-dimensional structure of the b subunit of RNA polymerase were
evaluated by aligning the rpoB sequences of Synechocystis sp.
strain PCC 6803 (NC000911) and Anabaena/Nostoc sp. strain PCC
7120 (BA000019) with the sequence of Thermus thermophilus
(YP_145079.1), for which the RNA polymerase structure has been
crystallized (2GHO) (Kuznedelov et al., 2002), by using RASWIN
Molecular Graphics v.2.6 (Sayle & Milner-White, 1995).
RESULTS AND DISCUSSION
Characterization of the indel within the rpoB gene
As described in Table S1, the PCR amplicons obtained for
12 of the 16 strains tested were 453 bp long, while shorter
products, 351 bp long, were obtained for the four
remaining strains: Planktothrix agardhii PCC 9214 and
Oscillatoria sp. strains PCC 8926, PCC 8954 and PCC 9631.
A multiple alignment showed that the longer rpoB
sequences display an indel of 120 nt and the shorter an
indel of 18 nt. The sequence of the inserts in the longer
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V. Gaget, S. Gribaldo and N. Tandeau de Marsac
Fig. 1. Alignment of amino acid sequences corresponding to the rpoB gene region that contains an indel. The rpoB gene
sequences available in public databases were selected and their corresponding amino acid sequences were aligned with the
sequences from 16 Planktothrix and Oscillatoria strains obtained in this study, and the Escherichia coli K-12 sequence. The
different cyanobacterial genera and strain identifiers are indicated on the left. The darker the highlighting of the amino acid, the
more conserved it is in all the aligned sequences.
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rpoB signature sequence for cyanobacterial typing
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V. Gaget, S. Gribaldo and N. Tandeau de Marsac
amplicons was used for a BLASTN search (Zhang & Madden,
1997; Zhang et al., 2000) against the NCBI non-redundant
(nr) sequence database. This insert sequence appeared to be
highly specific, appearing only in the rpoB gene of the two
Planktothrix strains NIVA CYA-126 and NIVA CYA-127.
Signature sequences in the rpoB genes of
different cyanobacterial taxa
To determine if cyanobacterial taxa other than Planktothrix
have inserts in their rpoB sequences, all available cyanobacterial rpoB sequence homologues were collected from
public databases. A multiple alignment of their corresponding amino acid sequences revealed the existence of a
conserved region containing inserts (indels) of lengths
varying from 6 to 44 aa (Fig. 1).
The indel is situated in the Rpb2_6 domain of RpoB
(determined in the Anabaena/Nostoc sp. strain PCC 7120
sequence) that is involved in DNA binding and DNA–
directed RNA polymerase activity (http://smart.emblheidelberg.de/). To locate the insert sequence in the
RpoB molecule more precisely, the three-dimensional
structure of the Synechocystis sp. strain PCC 6803 and
Anabaena/Nostoc sp. strain PCC 7120 RpoB proteins was
modelled based on an alignment of their sequences with
that of Thermus thermophilus using RASMOL v2.6 (Sayle &
Milner-White, 1995) (only the RpoB structure of
Synechocystis strain PCC 6803 is shown in Supplementary
Fig. S1, available with the online version of this paper).
This clearly showed that the indel sequence adds an extra
loop to a folding region of the Thermus thermophilus
consisting of b strands. The role of this loop is unknown,
but the presence of a few polar amino acids in its sequence
indicates that it is unlikely to interact with nucleic acids,
thus excluding a direct role in RNA polymerase function.
Moreover, no cationic binding sites are apparent. Peptide
binding to the loop sequence cannot be excluded but
further experiments are required to clarify this assumption.
RpoB signature sequences as a molecular tool for
cyanobacterial taxonomy
To assess the utility of these RpoB sequence signatures as a
tool for cyanobacterial taxonomy, we examined if the rpoB
gene followed a vertical inheritance or has been exchanged
among cyanobacterial strains by horizontal gene transfer.
For this, we compared phylogenies (built using three
methods: ML, NJ and MP) based on analysis of 16S rRNA
and rpoB gene sequence phylogenies of the same set of 134
strains. As the length of the rpoB indels is variable depending
on the genus (minimum 18 nt; maximum 132 nt), the
correction implemented by the Gblocks program removed
Fig. 2. ML tree based on 16S rRNA gene
sequences (1207 nt) from 134 cyanobacterial
strains for which an rpoB gene sequence is
available. Trees were rooted with the
Escherichia coli K-12 rpoB sequence as
outgroup. Roman numbers indicate clusters
of strains. Black triangles represent groups of
related strains; the number in each group is
indicated in parentheses. Bootstrap values are
shown for each node if .50 %. Bar, 0.05
substitutions per site.
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rpoB signature sequence for cyanobacterial typing
an important part of the sequence, leaving only 286 nt.
Consequently, due to the small size of the aligned sequences,
the rpoB tree appears less robust than the tree based on 16S
rRNA gene sequences (data not shown). However, the rpoB
tree is largely congruent with the 16S tree presented in Fig. 2
(five of the six clusters fully conserved). Moreover, when
aligned with the sequences of this rpoB region from other
closely related bacterial taxa (data not shown), cyanobacteria
form a well-separated group. These two observations show
there is no suggestion of obvious horizontal gene transfer,
indicating probable vertical inheritance.
Some of the clusters defined in the present study
correspond to the clades recently described by Gupta
(2009) using distinct indels located in different proteins.
Indeed, cluster I identified in this report corresponds to the
clade A described by Gupta, while cluster III corresponds
to clade C. Group II is an independent node in Gupta
(2009) and has not been included in a specific clade.
Strains in clusters IV, V and VI (or on independent nodes
for Synechocystis sp. PCC 6803 and Crocosphaera watsonii
WH 8501) group differently in clade B (Gupta, 2009), which
includes cyanobacteria from various taxa. This inconsistency
in the data obtained by the two research groups may be due
to a bias introduced by a difference in the number of strains
used in the two studies, a higher number being analysed in
the present report than in Gupta (2009).
To evaluate the usefulness of the rpoB signatures for
molecular typing of cyanobacterial strains, successive trees
(hereafter designated tier trees) were constructed for the
two clusters (V and VI) inferred from the 16S rRNA genebased tree (Fig. 2) for which the greater numbers of
nucleotide sequences (20 and 86, respectively) were
available. Starting from the general rpoB nucleotide
alignment, DNA sequences belonging to the same group
within clusters V (groups V-1 and V-2) and VI (groups
VI-1 and VI-2) were realigned separately and trees were
Fig. 3. (a) ML tree based on the rpoB nucleotide sequences of 86 cyanobacterial strains of cluster VI as defined in Fig. 2; (b) 37
strains of group VI-1; (c) Eight strains of subgroup VI-1-1; (d) 25 strains of subgroup VI-1-2. Groups and subgroups
correspond to those defined in Table 1. The number of nucleotides used to reconstruct the trees is indicated in parentheses.
Bootstrap values are shown for each node if .50 %. Bars, 0.05 substitutions per site.
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V. Gaget, S. Gribaldo and N. Tandeau de Marsac
constructed (Figs 3a and 4a). This made possible the
inclusion of residues that were discarded at the previous
level of analysis because of automatic gap removal. The
procedure was repeated on each new well-supported
subgroup (bootstrap value .50 %) until all the sequences
realigned possessed exactly the same signature sequence
and thus maximal resolution was achieved. For cluster V,
the number of nucleotide differences in the sequences
being limited, the maximal resolution was obtained after
two tier trees were constructed (data not shown), while for
cluster VI more tier trees were required (Figs 3 and 4).
Using this approach, we could deduce from the final
nucleotide sequences selected 80 distinct specific patterns
of RpoB protein signatures and 56 different indels (Table
1) whose clustering is congruent with the groups
established using the 16S rRNA gene and rpoB gene
sequences. Interestingly, these protein signatures differentiate strains at a subgenus level (Table 1). For example,
cluster VI can be divided into two groups (Figs 3a and 4a).
Group VI-1 contains members of the genera Nostoc and
Nodularia (Fig. 3b, c and d), while the group VI-2 contains
members of the genera Anabaena, Aphanizomenon and
Fig. 4. (a) ML tree based on the rpoB nucleotide sequences of 86 cyanobacterial strains of cluster VI as defined in Fig. 2; 49
strains of group VI-2 (b); 15 strains of subgroup VI-2-1 (c); 24 strains of subgroup VI-2-2 (d). Groups and subgroups
correspond to those defined in Table 1. The number of nucleotides used to reconstruct the trees is indicated in parentheses.
Bootstrap values are shown for each node if .50 %. Bars indicate 0.05 substitutions per site.
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rpoB signature sequence for cyanobacterial typing
Table 1. Patterns of RpoB signature sequences (including indels) in cyanobacteria
Cluster*/GroupD
SubgroupD
I
I-1
I-1-1
I-1-2
I-2
II
III
III-1
III-1-1
III-1-1-1
III-1-1-2
III-1-1-3
III-1-1-4
III-1-1-5
III-1-1-6
III-1-2
III-2
III-2-1
III-2-1-1
III-2-1-1a
III-2-1-1b
III-2-1-2
III-2-1-2a
III-2-1-2b
III-2-1-2c
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Common patternd
E(x)QAARDSGMV(x)VS(25-26x)Y(x)LQ
EAQAARDSGMVVVSRTNGVVTYVSADEIVVRPDDGGDPIVYRLQ
EAQAARDSGMVVVSRTSGVVTYVSADEIVVRPDDGGDPIVYRLQ
EGQAARDSGMVIVSDIDGAITYVSGEQIRVRGENGQEFAYPLQ
EPQAARDSGMVITSPVDGTISYVDATHIEVTADTGEKYGYALQ
E(x)QVARDSGMVPI(2x)VNG(x)V(2x)VDA(2x)I(x)V(4x)G(2x)H(x)H(x)LQ
E(x)QVARDSGMVPI(2x)VNG(x)V(x)YVDAN(x)IVV(4x)G(2x)H(x)H(x)LQ
E(x)QVARDSGMVPI(2x)VNG(x)V(x)YVDAN(x)IVV(4x)G(2x)H(x)H(x)LQ
ESQVARDSGMVPITKVNGIVSYVDANEIVVKDEDGNEHFHYLQ
ESQVARDSGMVPITKVNGTVSYVDANEIVVKDDHGNEHFHYLQ
ESQVARDSGMVPITKVNGIVSYVDANEIVVKDVDGNEHVHFLQ
ESQVARDSGMVPITKVNGIVSYVDANEIVVKDVDGNEHVHYLQ
ESQVARDSGMVPITKVNGTVSYVDANEIVVKGEDGNEHFHYLQ
ETQVARDSGMVPISQVNGTVTYVDANSIVVTDDEGGEHLHELQ
ETQVARDSGMVPISKVNGTVSYVDANAIVVTDDEGNDHTHYLQ
ETQVARDSGMVPI(x)RVNG(x)V(2x)VDA(x)AI(x)V(x)DE(x)G(2x)H(x)H(x)LQ
ETQVARDSGMVPISRVNG(x)V(2x)VDA(x)AI(x)V(x)DE(x)G(2x)H(x)H(x)LQ
ETQVARDSGMVPISRVNGTV(2x)VDA(x)AIVV(x)DE(x)G(2x)HTHFLQ
ETQVARDSGMVPISRVNGTVVYVDANAIVVLDEDGQEHTHFLQ
ETQVARDSGMVPISRVNGTVTFVDATAIVVRDEEGYDHTHFLQ
ETQVARDSGMVPISRVNG(x)V(x)FVDATAI(x)V(x)DE(x)G(2x)H(x)HYLQ
ETQVARDSGMVPISRVNGTVTFVDATAIVVRDEEGVDHSHYLQ
ETQVARDSGMVPISRVNGMVTFVDATAIIVRDEDGVDHTHYLQ
ETQVARDSGMVPISRVNGTVIFVDATAIVVQDEDGQEHTHYLQ
Species
Strain
Synechococcus sp.
JA23Ba
Synechococcus sp.
JA33Ab
Gloeobacter violaceus
PCC 7421
Synechococcus elongatus
PCC 6301; PCC 7942
Prochlorococcus marinus
MIT 9301
Prochlorococcus marinus
AS 9601
Prochlorococcus marinus
Prochlorococcus marinus
CCMP 1986
(5MED4)
MIT 9515
Prochlorococcus marinus
MIT 9312
Prochlorococcus marinus
CCMP 1375 (5SS120)
Prochlorococcus marinus
NATL1A; NATL2A
Synechococcus sp.
CC 9902
Synechococcus sp.
CC 9311
Synechococcus sp.
WH 7803
Prochlorococcus marinus
MIT 9303; MIT 9313
Synechococcus sp.
WH 8102
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V. Gaget, S. Gribaldo and N. Tandeau de Marsac
Table 1. cont.
Cluster*/GroupD
SubgroupD
III-2-2
III-2-3
IV
IV-1
IV-2
IV-3
V
V-1
V-1-1
V-1-2
V-2
V-2-1
V-2-2
V-2-3
V-2-4
VI
VI-1
VI-1-1
VI-1-1-1
VI-1-1-1a
VI-1-1-1b
VI-1-1-2
VI-1-1-2a
VI-1-1-2b
178
Common patternd
ETQVARDSGMVPISRVNGTVTYVDANAIVVQDEDGNDHTHFLQ
ETQVARDSGMVPITRVNGEVVFVDSTQIIVRDDQGVDHYHLLQ
EAQAARDSGMVIVSRTNGVVS(x)VDANRIR(x/-)KVAD(x)DK(x)IFGKSEIEYEIQ
EAQAARDSGMVIVSRTNGVVSYVDANRIRIKVADEDKDIFGKSEIEYEIQ
EAQAARDSGMVIVSRTNGVVSYVDANRIRIKVADEDKEIFGKSEIEYEIQ
EAQAARDSGMVIVSRTNGVVSHVDANRIRKVADQDKEIFGKSEIEYEIQ
EAQAARDSGMVIVS(2x)DGE(x)SY(x)DG(13-47x)EYELQ
EAQAARDSGMVIVSQIDGEVSYVDGARIRVTSPEGQEVEYELQ
EAQAARDSGMVIVSRTDGEVSYIDGSCIRVMDTTGKEHEYELQ
EAQAARDSGMVIVSRMDGEISYIDGSRIIVKSLTVDADSNSSEESFLIREYDDLDLKTKQPSVWKQKYAGYIEYELQ
EAQAARDSGMVIVSRMDGEVSYIDGSRIIVKTLGVDAEPNSSEESFLIREYDDLDLKTKQPSVWKQKYAGYIEYELQ
EAQAARDSGMVIVSRTDGEVSYIDGARIIVKSLAADAESNFSEESFLIREYDDLDLKTKQPSVWKQKYAAYIEYELQ
EAQAARDSGMVIVSRTDGEVSYIDGSCIRVIDNNGKEYEYELQ
(x)AQGARDSGMV(x)VSRTD(2x)V(x)YVDA(2x)IRVR(9-46x)LS
(x)AQGARDSGMV(x)VSRTDGDVTYDVATEIRVRPK(9-17x)LS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPK(4x)E(2x)Y(x)LS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPK(4x)E(x)KY(x)LS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKPNASELKYYLS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKTSTQEIKYRLS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKPNTSEIRY(x)LS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKPNTSEIRYLLS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKPNTSEIRYTLS
Species
Strain
Synechococcus sp.
CC 9605
Synechococcus sp.
RCC 307
Microcystis sp.
130
Microcystis sp.
NIES 843§; 269
Microcystis aeruginosa
PCC 7806
Lyngbya sp.
PCC 8106
Oscillatoria sp.
PCC 8926; PCC 8954;
PCC 9631
PCC 9637; PCC 9702;
PCC 7805; PCC 9239
Planktothrix agardhii
Oscillatoria sp.
PCC 8927
Oscillatoria sp.
PCC 9018
Planktothrix agardhii
PCC 9214
Nostoc ellipsosporum
V
Nostoc sp.
152
Nostoc muscorum
I (5Lukesova 2/91); II
Nostoc calcicola
III; VI (5VI.5)
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rpoB signature sequence for cyanobacterial typing
Table 1. cont.
Cluster*/GroupD
SubgroupD
VI-1-2
VI-1-2-1
VI-1-2-1a
VI-1-2-1b
VI-1-2-1c
VI-1-2-1d
VI-1-2-2
VI-1-2-2a
VI-1-2-2b
VI-1-2-2c
VI-1-2-3
Ungrouped
VI-2
VI-2-1
VI-2-1-1
VI-2-1-1a
VI-2-1-1b
VI-2-1-1c
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Common patternd
(x)AQGARDSGMV(x)VSRTDGDVTYVDATEIRVRPK(6-14x)RYRLS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPK(4-12x)EIRYRLS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKEGNSEIRYRLS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKDNNSGESERSRNEIRYRLS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKDNNSGDSERSRNEIRYRLS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKDSNSGEPERSRNEIRYRLS
(x)AQGARDSGMVVVSRTDGDVTYVDATEIRVRPKGGN(3x)RYRLS
EAQGARDSGMVVVSRTDGDVTYVDATEIRVRPKGGNSEIRYRLS
EAQGARDSGMVVVSRTDGDVTYVDATEIRVRPKGGNSETRYRLS
KAQGARDSGMVVVSRTDGDVTYVDATEIRVRPKGGNFRIRYRLS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKDGNSEIRYRLS
EAQGARDSGMVIVSRTDGDVVYVDATEIRVRVSGQLPAASGKSTDNGQLTSQKGQEIRYTVS
EAQGARDSGMVIVSRTDGDVVYVDATEIRVRVSGQLPTASGKSTDNGQLTSQKGQEIRYTVS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRPKPNSPEIRYTVS
EAQGARDSGMVIVSRTDGDVTYVDATEIRVRVSNQSKGSEHGQATNQKPQEIRVYYLS
EAQ(x)ARDSGMVIVSRT(x)G(x)V(x)YVDA(2x)IRVR(21-46x)Y(x)LS
EAQAARDSGMVIVSRTDGEVVYVDATEIRVRV(20-33x)Y(x)LS
EAQAARDSGMVIVSRTDGEVVYVDATEIRVRVSS(x)WSV(x)SGQSSLAEKR(x)TDNEQL(3x)K(x)Q(x)IRYNLS
EAQAARDSGMVIVSRTDGEVVYVDATEIRVRVSSQWSVISGQSSLAEKRTTDNEQLATDKPQDIRYNLS
EAQAARDSGMVIVSRTDGEVVYVDATEIRVRVSSEWSVVSGQSSLAEKRSTDNEQLMSEKFQEIRYNLS
EAQAARDSGMVIVSRTDGEVVYVDATEIRVRVSSQWSVISGQSSLAEKRTTDNEQLTTDKPQEIRYNLS
Species
Strain
Nodularia harveyana
Huebel 1983/300
Nodularia harveyana
BECID 29
Nodularia harveyana
BECID 27
Nodularia harveyana
Bo53
Nodularia sphaerocarpa
Nodularia sphaerocarpa
PCC 7804; BECID 35;
BECID 36
HKVV; Up16a; Up16f
Nodularia spumigena
Fae19
Nodularia spumigena
Anabaena variabilis
PCC 9350; HEM;
AV63; Huebel 1987/
311; AV1
ATCC 29413
Anabaena sp.
PCC 7120
Trichormus azollae
Kom BAI 1983
Trichormus dololium
Dololium 1
Anabaena planctonica
1tu36s8
Anabaena planctonica
1tu33s8; 1tu28s8;
1tu33s10; 1tu30s13
Anabaena spiroides
1tu39s17
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V. Gaget, S. Gribaldo and N. Tandeau de Marsac
Table 1. cont.
Cluster*/GroupD
SubgroupD
VI-2-1-2
VI-2-2
VI-2-2-1
VI-2-2-2
VI-2-3
VI-2-3-1
VI-2-3-2
VI-2-4
VI-2-4-1
VI-2-4-2
Ungrouped
Unclustered
180
Common patternd
EAQAARDSGMVIVSRTDGDVVYVDATEIRVRVGGQRLVVGDEEKTGDREQLRTDKPQELKYKLS
EAQAARDSGMVIVSRTDGDV(x)YVDATEIRVRASGQLSAASGS(67x)Q(x)IEKGQELKYKLS
EAQAARDSGMVIVSRTDGDVVYVDATEIRVRASGQLSAASGSQVIEKGQELKYKLS
EAQAARDSGMVIVSRTDGDVIYVDATEIRVRASGQLSAASGSPQPIEKGQELKYKLS
EAQGARDSGMVIVSRTDGDVVYVDAAEIRVRVREQEKED(23x)RGQDTEKNVKLGDTF(2x)SPRLPITSSSS(x)PKE(x)RYVLS
EAQGARDSGMVIVSRTDGDVVYVDAAEIRVRVREQEKEDTLTRGQGDTEKNVKLGDTFTASPRLPITSSSSPPKEIRYVLS
EAQGARDSGMVIVSRTDGDVVYVDAAEIRVRVREQEKEDHGRGQDTEKNVKLGDTFSPSPRLPITSSSSSPKEVRYVLS
EAQAARDSGMVIVSRTHGDVVYVDATEIRVRVSGQ(x)SAASGSQVIEKGQEIRY(x)LS
EAQAARDSGMVIVSRTHGDVVYVDATEIRVRVSGQLSAASGSQVIEKGQEIRYNLS
EAQAARDSGMVIVSRTHGDVVYVDATEIRVRVSGQISAASGSQVIEKGQEIRYTLS
EAQGARDSGMVIVSRTDGDVVYVDAAQIRVRVRGDLSGVTGNLIAGKQHTNEQGQQVTDREIRYVLS
EAQGARDSGMVIVSRTDGDVVYVDAAEIRVRVREQTTDKTLTQSPASSKESPTGKPQEVTYYLS
EAQAARDSGMVIVSRTDGEVVYVDATEIRVRVSGQSLLANQQTTDKEQHAPEKPQEIKYTLS
EAQAARDSGMVIVSRTDGEVVYVDATEIRVRVSSQWSIVSGQQTTDNEQQTTDKPQEIKYTLS
EAQGARDSGMVIVSRTDGDVTYVDAAEIRVRVREPLTETNTQHPPKEVRYVLS
EAQAARDSGMVIVSRTEGIVTYADANEIRVKVTGPEGHEKVGQEIQYILQ
EAQAARDSGMVIVSRTHGIVTYVDATEIRVQPHSPDNPAEKGEEIVYPIQ
EAQAARDSGMVILSQTNGVVSYVDANQIRVKTDNGPEITYTLQ
Species
Strain
Aphanizomenon flos-aquae
1tu26s2; 1tu29s19;
1tu37s13
Anabaena sp.
90
Anabaena circinalis
1tu34s5
Trichormus variabilis
HINDAK 2001/4
Trichormus variabilis
GREIFSWALD
Anabaena oscillarioides
BECID 22; BECID 32
Anabaena cylindrica
XP6B
Anabaena oscillarioides
BO HINDAK 1984/43
Anabaena augstumalis
SCHMIDKE
JAHNKE/4a
Anabaena sp.
1tu34S7
Aphanizomenon issatschenkoi
0tu37S7
Anabaena sp.
PCC 7108
Crocosphaera watsonii
WH8501
Synechocystis sp.
PCC 6803
Thermosynechococcus elongatus
BP1
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rpoB signature sequence for cyanobacterial typing
Table 1. cont.
Cluster*/GroupD
SubgroupD
Cyanobacteria
Common patternd
Species
EPQAARDSGMVVVTRTDGEVSYVDSTKISVIDKEGNQADYPLCKYQ
(2x)Q(x)ARDSGMV(30-72x)
Trichodesmium erythraeum
Strain
IMS 101
*Clusters have been determined according to the 16S rRNA gene sequence-based phylogenetic tree presented in Fig. 2, and specified into groups
depending on their organization in rpoB trees.
DGroups and subgroups have been determined based on signature sequence identity.
dIndels are in shown in bold; signature sequences are underlined.
§Strain NIES 843 has been reassigned to the species Microcystis aeruginosa.
Trichormus (Fig. 4b, c and d). In group VI-1, the Nostoc
and Nodularia strains are separated into two monophyletic
subgroups (VI-1-1 and VI-1-2) (Fig. 3c and d). Moreover,
subgroups VI-1-2-1 and VI-1-2-3 include strains with the
same species attribute, Nodularia harveyana and Nodularia
spumigena, respectively. This shows that, in some cases, the
signature can differentiate strains at the species level.
However, some other subgroups contain two different
species, e.g. subgroup VI-1-2-2b includes both Nodularia
sphaerocarpa and Nodularia spumigena (Table 1 and Fig.
3d). Although one cannot exclude that the mutation rate of
this rpoB region differs in some strains, this might result
from a misleading species assignment.
Rajaniemi et al. (2005) published a partial tree from the
same rpoB gene sequence portion, but did not distinguish
between groups VI-1 and VI-2, although groups A and B
defined by these authors appear to perfectly correspond to
the subgroups VI-2-1-1 and VI-2-1-2 identified in the
present study (Fig. 4b and c; Table 1). In this example, even
if the signature sequence comprises a small number of
nucleotides, it provides the same resolution as longer
sequences. The fact that strains belonging to the genera
Anabaena, Aphanizomenon and Trichormus are not separated into discrete subgroups (Fig. 4c and d) supports the
hypothesis that these cyanobacterial strains might belong
to the same genus, within which several species can be
distinguished, as discussed by Rajaniemi et al. (2005).
Interestingly, in cluster V (Table 1), RpoB signature
sequences can be useful to identify groups first on the
basis of pattern length and second on the basis of their
specific amino acid sequence. For example, in cluster V, the
Oscillatoria strains present six and 40 aa indels. Notably, all
the Planktothrix strains (P. agardhii and P. rubescens)
whose sequences have been determined in the present
study fall within group V-2 with a longer indel, with the
exception of strain PCC 9214 (Table 1). Thus, knowing
that Planktothrix strains can be distinguished on the basis
of the rpoB signature sequence length, a simple PCR might
be sufficient, as a primary screen, to identify subgroups to
which particular strains belong. In contrast, the five
Oscillatoria strains analysed in the present study are
distributed in three subgroups, V-1-2, V-2-2 and V-2-3,
that include either long or short sequences (Table 1). This
http://ijs.sgmjournals.org
observation suggests that a re-examination of the taxonomic position of Oscillatoria sp. strains PCC 8926, PCC
8927, PCC 8954, PCC 9018 and PCC 9631, as well as
Planktothrix agardhii PCC 9214, using a polyphasic
approach (Suda et al., 2002) is required to clearly establish
if they belong to the genera Planktothrix or Oscillatoria.
Concluding remarks
The signatures of the rpoB sequences of cyanobacteria
identified in this study that allow the identification of
strains at a subgenus level even permit species separation
for some taxa. These signature sequences might thus be
particularly useful for the identification and classification
of a large number of isolates, as well as for assessment of
cyanobacterial biodiversity in natural microbial communities and for following the seasonal abundance of cyanobacteria in a given community over time. Generally, these
rpoB signatures may be used as a tool to change the current
view of the classification of nostocacean strains, and
possibly that of other cyanobacterial groups. The contribution of molecular typing provided by rpoB and other
housekeeping gene indels may well advance the resolution
of specific cases in cyanobacterial taxonomy or provide an
intermediate level of variability suited to various molecular
typing applications, including DGGE and SSCP (Boutte
et al., 2006; Dorigo et al., 2005).
ACKNOWLEDGEMENTS
We wish to thank the CAE (Centre d’Analyses Environnementales) of
Veolia Environment, the Institut Pasteur and CNRS URA-2172 for
funding this work. We are grateful to the CAE of Veolia Environment
for providing a thesis fellowship to V. G., and to K. Delabre and
F. Enguehard for constant support during this study. We express our
gratitude to T. Rose for his advice on three-dimensional predictions
and thank J. P. Rasmussen for helpful comments and his revision of
the English during the preparation of this paper.
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